Measurement of a fluorescent analyte using tissue excitation

ABSTRACT

An apparatus and method for noninvasive measurement of a fluorescent analyte concentration in the blood of a patient by exciting the blood and the analyte at two wavelength ranges and measuring the emission spectrum of the fluorescent analyte when (i) the difference of emission intensities at the excitation wavelength ranges of the fluorescent analyte is greater than that of background fluorophores, and (ii) when blood absorbance at the two excitation wavelength ranges is similar. An apparatus and method for measurement of a fluorescent analyte concentration in the blood of a patient is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application61/535,064, filed Sep. 15, 2011, which is incorporated by references inits entirety herein.

TECHNICAL FIELD

An apparatus and method for measurement of one or more fluorescentanalyte concentration(s) in the blood of a patient by exciting the bloodand the analyte at two wavelengths. More particularly, the apparatus andmethod measure the concentration of erythrocyte zinc protoporphyrin(referred to herein as “eZnPP” or “ZnPP”) and erythrocyte protoporphyrinIX (referred to herein as “ePP” or “PP”) in the red blood cells of apatient.

BACKGROUND

Iron deficiency remains the most common form of malnutrition worldwide,increasing the risk of disability and death among more than two billionpeople. Lack of iron causes anemia, decreases physical capabilities,impairs cognitive and behavioral development, compromises immuneresponsiveness and when severe, increases mortality during infancy andchildhood. Iron supplements are needed for prevention of iron deficiencyin those with increased iron requirements, especially infants, childrenand women of childbearing age, and for correction of iron deficiencyanemia in all affected individuals.

However, in areas with endemic malaria, untargeted iron supplementationis no longer recommended as a means of providing additional iron becausean increased risk of hospitalization and death was found in a trial ofuniversal iron and folic acid supplementation for preschool children inPemba, Tanzania. Using an elevated eZnPP/heme molar ratio (>80 μmol/molheme) as the criterion for iron deficiency, iron-deficient children werefound to benefit from supplementation. Their risk of severe illness anddeath decreased by 38%. In contrast, iron-replete children were harmedby supplementation. In fact, their risk of severe illness and deathincreased by 63% after iron supplementation. See, e.g., Sazawal S. etal., Effects of routine prophylactic supplementation with iron and folicacid on admission to hospital and mortality in preschool children in ahigh malaria transmission setting: community-based, randomised,placebo-controlled trial. Lancet 2006; 367: 133-143. In view of thisrisk, a World Health Organization (WHO) Consultation recommended that,in malaria-endemic areas, (i) iron supplements should be given tochildren only after screening for iron deficiency and (ii) themeasurement of eZnPP was the preferred indicator for identifyingiron-deficient children who could benefit from iron supplementation.See, WHO Conclusions and recommendations of the WHO Consultation onprevention and control of iron deficiency in infants and young childrenin malaria-endemic areas. Food Nutr Bull 2007; 28: S621-7.

In resource-limited settings, like those in regions with endemicmalaria, the use of the existing front-face hematofluorometer techniquefor measurement of eZnPP is constrained by the requirement for a bloodsample obtained by finger- or venipuncture, the necessity for a trainedtechnician for operation, use of an electrical power supply, a need forfrequent recalibration and expense. Other currently available means ofassessing iron status also require blood samples and even more complexand costly laboratory facilities and processing. Because of the lack ofmeans to determine iron status, the effective result of the WHOrecommendation has been the cessation of programs of ironsupplementation in almost all malarial areas.

Thus, there is a need for a new technique that overcomes the technicaldifficulties of existing invasive techniques for identifying thoseindividuals in malarial areas who would benefit from ironsupplementation to permit safe and effective prevention and correctionof iron deficiency, while avoiding harm to those who are iron replete.

Globally, 30% to 70% of the populations in developing countries are irondeficient, with the highest prevalence among persons who have diets lowin bio-available iron. In developed countries, despite increased amountsof dietary bio-available iron, iron nutrition nevertheless remains aproblem in subpopulations with the highest iron requirements, especiallyamong infants, children and women of childbearing age. Without ironsupplementation, most women will become iron deficient during pregnancy.Thus, screening for iron deficiency is a crucial component ofhealthcare. Initially, iron deficiency may be asymptomatic or produceonly nonspecific manifestations, such as weakness and easy fatigability.As iron deficiency becomes more severe, anemia develops andprogressively restricts work capacity and tolerance of physicalexertion. Early detection of iron deficiency permits prompt recognitionand management of underlying causes. Most commonly, a diet withinadequate amounts of bio-available iron is responsible. In theseindividuals, iron deficiency may be corrected by nutritional approaches,such as consuming iron-rich food as well as food which helps the bodyabsorb iron more effectively, such as food high in vitamin C, or by ironsupplementation.

Thus, there is also a need for periodic iron monitoring in a safe,effective manner without the need for a blood sample. There is also aneed to provide a technique and apparatus that can be used as apoint-of-care screening device for iron deficiency in pediatric,obstetric and medical facilities, and in blood donation centersworldwide, and by individuals to monitor their own iron status in theirhomes or portably, without the need to be in a clinical setting. BecauseeZnPP is also elevated in lead poisoning, a noninvasive method would beuseful for screening those people at risk from occupational orenvironmental exposure.

There is also a need to provide a technique and apparatus for measuringthe concentration of an analyte in the blood of a patient. For example,in certain settings—such as a hospital or clinical environment withaccess to sterile conditions and adequate equipment—it is acceptableand/or desirable to analyze iron levels in the blood. A technique andapparatus which provides greater accuracy and consistency when comparedwith existing techniques is needed.

SUMMARY

In one aspect, an apparatus for noninvasive measurement of aconcentration of one or more fluorescent analyte(s) in the blood of apatient is provided which includes a light source for providingexcitation of the analyte and the blood at a first wavelength range anda second wavelength range, the first and second excitation wavelengthranges selected such that the analyte exhibits a difference in emissionintensities at the first and second excitation wavelength ranges andsuch that light absorbance of blood at the first and second wavelengthranges is similar; one or more detectors for detecting a portion of theemission spectra of the fluorescent analyte at the first excitationwavelength range and the second excitation wavelength range; and aprocessor adapted to determine a derived signal representative of theconcentration of the analyte based on the difference between the portionof the emission spectra excited at the first excitation wavelength rangeand the second excitation wavelength range.

In some embodiments, the apparatus is for measurement of a concentrationof one or more fluorescent analyte(s) in whole blood.

In some embodiments, a tunable filter unit is provided which excites theblood and the analyte(s) at the first wavelength range and the secondwavelength range. In some embodiments, the tunable filter unit includesa first optical filter and a second optical filter, the first and secondoptical filters are capable of independent variation of the angle ofincidence of light provided by the light source. In some embodiments,the tunable filter unit includes two tunable bandpass filters, (such asSemrock Versachrome® filters). In some embodiments, the tunable filterunit includes a first optical filter and a second optical filter and athird optical element to correct for offset of the light passing throughthe first and second optical filters.

In some embodiments, the apparatus further includes one or more opticalfilters, wherein the emission spectra of the fluorescent analyte definesa wavelength range, and wherein the detector includes one or more lightsensitive elements receiving light through the one or more opticalfilters transmitting light in the wavelength range of the emissionspectra of the fluorescent analyte.

In some embodiments, the emission spectra of the fluorescent analytedefine an emission maximum, wherein a first portion of the detectorreceives light through the optical filters transmitting light in thewavelength range of the emission spectra of the fluorescent analyte, andwherein a second portion of the detector receives light through opticalfilters transmitting light in a wavelength range outside the emissionmaximum of the fluorescent analyte.

In some embodiments, the light source is a lamp, one or more laserdiodes, or one or more light emitting diodes.

In some embodiments, the apparatus further includes an optical fiberassociated with the light source. In some embodiments, the apparatusfurther includes an optical fiber associated with the detector.

In some embodiments, the apparatus further includes a probe including anoptical fiber associated with the light source and an optical fiberassociated with the detector. In some embodiments, the probe includes aplurality of optical fibers associated with the light source surroundingan optical fiber associated with the detector.

In some embodiments, the interfiber spacing of the optical fiberassociated with the light source and the optical fiber associated withthe detector is selected such that the derived signal is insensitive tothe blood volume fraction.

In some embodiments, the interfiber spacing of the optical fiberassociated with the light source and the optical fiber associated withthe detector is selected to achieve a maximum detection sensitivity at aselected depth of the tissue. In some embodiments, the selected depth ofthe tissue is selected as the depth having the highest expectedconcentration of the fluorescent analyte.

In some embodiments, the apparatus further includes a power source. Insome embodiments, the power source is a rechargeable battery.

In some embodiments, the apparatus includes a housing adapted to receivethe detector, the processor and the power source. In some embodiments,the housing is less than 6 inches in length.

In some embodiments, the apparatus includes an output component. In someembodiments, the apparatus includes a housing adapted to receive thedetector, the processor and the output component. In some embodiments,the apparatus the output component is a display screen, a speaker or avibrator.

In some embodiments, the output component provides an indication of theconcentration of analyte in the tissue. In some embodiments, the outputcomponent provides an indication that the concentration of analyteexceeds a predetermined threshold.

In some embodiments, the apparatus includes memory storing at least oneprevious concentration of analyte, and wherein the output component isadapted to provide an indication that the concentration of analyte isincreasing or decreasing from the previous concentration of analyte.

In some embodiments, the apparatus includes a communications component.In some embodiments, the communications component comprises an RFtransmitter, a USB connector, an IR transmitter, a cellular phone or aWiFi transmitter.

A system for noninvasive measurement of a concentration of a fluorescentanalyte in the blood of a patient is provided that includes theapparatus described hereinabove, and a monitor unit, wherein thecommunications component provides an output signal relating to theconcentration of analyte to the monitor unit.

In some embodiments, the processor is adapted to provide the outputsignal to the monitor unit when the analyte concentration exceeds apredetermined threshold.

In some embodiments, the monitor unit comprises a user interface. Insome embodiments, the user interface provides an indication of theconcentration of analyte in the tissue. In some embodiments, the userinterface provides an indication that the concentration of analyteexceeds a predetermined threshold. In some embodiments, the monitor unitcomprises memory storing at least one previous concentration of analyte,and wherein the user interface provides an indication that theconcentration of analyte is increasing or decreasing from the previousconcentration of analyte. In some embodiments, the user interfaceprovides the user with the option of storing a health goal related tothe concentration of analyte, and wherein the user interface provides anindication of a trend towards or away from the health goal.

In some embodiments, the user interface provides a treatment suggestionrelated to the concentration of analyte. In some embodiments, thetreatment suggestion comprises ingestion of a quantity of nutrition. Insome embodiments, the treatment suggestion comprises administration of aquantity of a pharmaceutical compound.

In some embodiments, the monitor unit is portable. In some embodiments,the monitor unit is a personal computer. In some embodiments, themonitor unit is a telephone.

An apparatus for noninvasive measurement of a concentration oferythrocyte zinc protoporphyrin (eZnPP) as the eZnPP/heme ratio in theblood of a patient is provided which includes a light source forproviding excitation of the tissue at a first wavelength range and asecond wavelength range, the first excitation wavelength range selectedat the excitation peak of eZnPP and the second excitation wavelengthrange selected so that the absorbance of blood is similar to that of thefirst excitation wavelength range; one or more detectors for detecting aportion of the emission spectra at the first excitation wavelength rangeand the second excitation wavelength range; and a processor fordetermining the concentration of eZnPP based on the difference betweenthe portion of the emission spectra excited at the first excitationwavelength range and the second excitation wavelength range.

In some embodiments, the apparatus is for measurement of a concentrationof eZnPP in whole blood.

In some embodiments, a tunable filter unit is provided which excites theblood and eZnPP at the first wavelength range and the second wavelengthrange. In some embodiments, the tunable filter unit includes a firstoptical filter and a second optical filter, the first and second opticalfilters capable of independent variation of the angle of incidence oflight provided by the light source. In some embodiments, the tunablefilter unit includes two tunable bandpass filters, (such as SemrockVersachrome® filters). In some embodiments, the tunable filter unitincludes a first optical filter and a second optical filter and a thirdoptical element to correct for offset of the light passing through thefirst and second optical filters.

In some embodiments, the apparatus further includes one or more opticalfilters, wherein the emission spectra of eZnPP defines a wavelengthrange, and wherein the detector includes one or more light sensitiveelements receiving light through the one or more optical filterstransmitting light in the wavelength range of the emission spectra ofeZnPP.

In some embodiments, the emission spectra of eZnPP define an emissionmaximum, wherein a first portion of the detector receives light throughthe optical filters transmitting light in the wavelength range of theemission spectra of eZnPP, and wherein a second portion of the detectorreceives light through optical filters transmitting light in awavelength range outside the emission maximum of eZnPP.

In some embodiments, the light source is a lamp, one or more laserdiodes, or one or more light emitting diodes.

In some embodiments, the apparatus further includes an optical fiberassociated with the light source. In some embodiments, the apparatusfurther includes an optical fiber associated with the detector.

In some embodiments, the apparatus further includes a probe including anoptical fiber associated with the light source and an optical fiberassociated with the detector. In some embodiments, the probe includes aplurality of optical fibers associated with the light source surroundingan optical fiber associated with the detector.

In some embodiments, the interfiber spacing of the optical fiberassociated with the light source and the optical fiber associated withthe detector is selected such that the derived signal is insensitive tothe blood volume fraction.

In some embodiments, the interfiber spacing of the optical fiberassociated with the light source and the optical fiber associated withthe detector is selected to achieve a maximum detection sensitivity at aselected depth of the tissue. In some embodiments, the selected depth ofthe tissue is selected as the depth having the highest expectedconcentration of eZnPP.

In some embodiments, the apparatus further includes a power source. Insome embodiments, the power source is a rechargeable battery.

In some embodiments, the apparatus includes a housing adapted to receivethe detector, the processor and the power source. In some embodiments,the housing is less than 6 inches in length.

In some embodiments, the apparatus includes an output component. In someembodiments, the apparatus includes a housing adapted to receive thedetector, the processor and the output component. In some embodiments,the output component is a display screen, a speaker or a vibrator.

In some embodiments, the output component provides an indication of theconcentration of eZnPP in the tissue. In some embodiments, the outputcomponent provides an indication that the concentration of eZnPP exceedsa predetermined threshold.

In some embodiments, the apparatus includes memory storing at least oneprevious concentration of eZnPP, and wherein the output component isadapted to provide an indication that the concentration of eZnPP isincreasing or decreasing from the previous concentration of analyte.

In some embodiments, the apparatus includes a communications component.In some embodiments, the communications component comprises an RFtransmitter, a USB connector, an IR transmitter, a cellular phone or aWiFi transmitter.

A system for noninvasive measurement of a concentration of eZnPP in theblood of a patient is provided which includes the apparatus describedhereinabove, and a monitor unit, wherein the communications componentprovides an output signal relating to the concentration of eZnPP to themonitor unit.

In some embodiments, the processor is adapted to provide the outputsignal to the monitor unit when the eZnPP concentration exceeds apredetermined threshold.

In some embodiments, the monitor unit comprises a user interface. Insome embodiments, the user interface provides an indication of theconcentration of eZnPP in the tissue. In some embodiments, the userinterface provides an indication that the concentration of eZnPP exceedsa predetermined threshold. In some embodiments, the monitor unitcomprises memory storing at least one previous concentration of eZnPP,and wherein the user interface provides an indication that theconcentration of eZnPP is increasing or decreasing from the previousconcentration of eZnPP. In some embodiments, the user interface providesthe user with the option of storing a health goal related to theconcentration of eZnPP, and wherein the user interface provides anindication of a trend towards or away from the health goal.

In some embodiments, the user interface provides a treatment suggestionrelated to the concentration of eZnPP. In some embodiments, thetreatment suggestion comprises ingestion of a quantity of nutrition. Insome embodiments, the treatment suggestion comprises administration of aquantity of a pharmaceutical compound.

In some embodiments, the monitor unit is portable. In some embodiments,the monitor unit is a personal computer. In some embodiments, themonitor unit is a telephone.

An apparatus for simultaneous measurement of a concentration oferythrocyte zinc protoporphyrin (eZnPP) and erythrocyte protoporphyrinIX (ePP) as the eZnPP/heme ratio and ePP/heme ratio in the blood of apatient is provided including a light source for providing excitation ofthe tissue at a first wavelength range and a second wavelength range,the first excitation wavelength range selected at the excitation peak ofeZnPP and the second excitation wavelength range selected so that theabsorbance of blood is similar to that of the first excitationwavelength range; one or more detectors for detecting a portion of theemission spectra at the first excitation wavelength range and the secondexcitation wavelength range; and a processor for determining theconcentration of eZnPP and ePP based on the difference between theportion of the emission spectra excited at the first excitationwavelength range and the second excitation wavelength range.

An apparatus for noninvasive measurement of a concentration oferythrocyte zinc protoporphyrin (eZnPP) as the eZnPP/heme ratio in theblood of a patient is provided including a light source for providingexcitation of the tissue at about 425 nm and about 407 nm; a detectorfor detecting a portion of the emission spectra excited at about 425 nmand about 407 nm; and a processor for determining the concentration ofeZnPP based on the difference between the portion of the emissionspectra excited at about 425 nm and about 407 nm.

An apparatus for measurement of a concentration of erythrocyte zincprotoporphyrin (eZnPP) as the eZnPP/heme ratio in the blood of a patientis provided including a light source for providing excitation of thetissue at about 425 nm and about 407 nm; a detector for detecting aportion of the emission spectra excited at about 425 nm and about 407nm; and a processor for determining the concentration of eZnPP based onthe difference between the portion of the emission spectra excited atabout 425 nm and about 407 nm.

A method for noninvasive measurement of a concentration of a fluorescentanalyte in the blood of a patient is provided including exciting thetissue at a first wavelength range and a second wavelength range, thefirst and second excitation wavelength ranges selected such that thefluorescent analyte exhibits a difference in emission intensities at thefirst and second excitation wavelength ranges that is greater than thatof background fluorophores and light absorbance by blood at the firstand second excitation wavelength ranges is similar; detecting a portionof the emission spectra at the first excitation wavelength range and thesecond excitation wavelength range; and determining the concentration ofthe fluorescent analyte based on the difference between the emissionspectra excited at the first excitation wavelength range and the secondexcitation wavelength range.

An apparatus for filtering a beam of light is provided including a firstoptical filter defining an adjustable angle of incidence with respect tothe beam of light; a second optical filter defining an adjustable angleof incidence with respect to the beam of light; wherein the angle ofincidence of the first optical filter and the angle of incidence of thesecond optical filter are independently adjustable; wherein a centralwavelength of light passing through first and second optical filters istunable by adjustment of the angle of incidence of the first filter withrespect to the beam of light; and wherein the spectral bandwidth oflight passing through the first and second optical filters is tunable byadjustment of the angle of incidence of the first filter and the secondfilter with respect to the beam of light. In some embodiments, the firstand second optical filters comprise two tunable bandpass filters, (suchas Semrock Versachrome® filters). In some embodiments, a third opticalelement is provided to correct for offset of the light passing throughthe first and second optical filters.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a schematic view of an apparatus in accordance with anexemplary embodiment of the subject matter described herein.

Figure lA is a simplified schematic view of an apparatus in accordancewith an exemplary embodiment of the subject matter described herein.

FIG. 1B is a simplified schematic view of an apparatus, indicating afirst spacing between an excitation fiber and a detection fiber, inaccordance with an exemplary embodiment of the subject matter describedherein.

FIG. 1C is a simplified schematic view of an apparatus, indicating asecond spacing between an excitation fiber and a detection fiber, inaccordance with an exemplary embodiment of the subject matter describedherein.

FIG. 2 is a view of an apparatus in a free-beam instrumentalconfiguration in accordance with another exemplary embodiment of thesubject matter described herein, adapted for measurement of patientblood samples or tissue.

FIG. 3 illustrates the excitation spectra of eZnPP bound tooxyhemoglobin.

FIG. 4 illustrates the emission spectra of eZnPP bound to oxyhemoglobin.

FIG. 5 is a perspective view of a fiber optic probe head in accordancewith an exemplary embodiment of the subject matter described herein.

FIG. 6 is a cross-sectional view of tissue receiving the stimulationfrom the light source of an exemplary apparatus as disclosed in thesubject matter herein.

FIG. 7 illustrates the tissue autofluorescence spectrum from oral mucosaobtained using an exemplary apparatus as disclosed in the subject matterherein.

FIG. 8 illustrates the normalized blood absorbance and eZnPP excitationspectrum.

FIG. 9 is a perspective view of an apparatus and tissue phantom inaccordance with an exemplary embodiment of the subject matter describedherein.

FIG. 10 illustrates the excitation spectra for collagen with and withoutblood.

FIG. 11 illustrates the emission spectra for the simple tissue phantomof the oral mucosa.

FIG. 12 illustrates the emission spectra for the difference spectrum foralternating two-wavelength fluorescence excitation.

FIG. 13 illustrates a comparison of the output of an apparatus inaccordance with an exemplary embodiment shown in FIG. 2 (on the verticalaxis) with those provided by an Aviv hematofluorometer (on thehorizontal axis) in measurements of the eZnPP/heme ratio on a series ofpatient blood samples.

FIG. 14 is a side view of another apparatus in accordance with anexemplary embodiment of the subject matter described herein.

FIG. 15 is a side view of a further apparatus in accordance with anexemplary embodiment of the subject matter described herein.

FIG. 16 is a schematic view of an apparatus in accordance with anexemplary embodiment of the subject matter described herein.

FIG. 17 is a view of an apparatus in a free-beam instrumentalconfiguration in accordance with another exemplary embodiment of thesubject matter described herein, adapted for measurement of patientblood samples or tissue.

FIG. 18 is a view of a portion of an apparatus in accordance withanother exemplary embodiment of the subject matter described herein.

FIG. 19 is a schematic view of the apparatus of FIG. 18 in accordancewith an exemplary embodiment of the subject matter described herein.

FIG. 20 illustrates the transmission of light through the apparatus ofFIG. 18 in accordance with an exemplary embodiment of the subject matterdescribed herein.

FIG. 21 is a schematic view of a portion of an apparatus in accordancewith an exemplary embodiment of the subject matter described herein.

FIGS. 22-23 are schematic views of a portion of an apparatus inaccordance with an exemplary embodiment of the subject matter describedherein.

FIG. 24 is a schematic view of a portion of an apparatus in accordancewith an exemplary embodiment of the subject matter described herein.

FIGS. 25-30 illustrate the excitation and emissions spectra of tissuemeasured in accordance with an exemplary embodiment of the subjectmatter described herein.

FIGS. 31-36 illustrate a comparison of conventional techniques with theresults obtained in accordance with an exemplary embodiment of thesubject matter described herein.

FIG. 37 illustrates an emission spectrum in accordance with an exemplaryembodiment of the subject matter described herein.

FIG. 38 illustrates an emission spectrum in accordance with an exemplaryembodiment of the subject matter described herein.

FIG. 39 illustrates a difference spectrum in accordance with anexemplary embodiment of the subject matter described herein.

FIGS. 40-41 illustrate a correlation between the eZnPP/heme ratioobtained by a hematofluorometer and by HPLC.

FIGS. 42-43 illustrate a correlation between the measured fluorescenceintensity at 593 nm evaluated by HPLC and a method in accordance with anexemplary embodiment of the subject matter described herein.

FIGS. 44-45 illustrate a correlation between the measured fluorescenceintensity of the difference spectrum at 593 nm evaluated by HPLC and amethod in accordance with an exemplary embodiment of the subject matterdescribed herein.

FIGS. 46-47 illustrate a correlation between the measured fluorescenceintensity of the difference spectrum at 627 nm evaluated by a method inaccordance with an exemplary embodiment of the subject matter describedherein and the PP/heme ratio measured by HPLC.

FIG. 48 illustrates the eZnPP/PP ratio calculated from the eZnPP and PPfluorescence intensities as evaluated by a method in accordance with anexemplary embodiment of the subject matter described herein.

DETAILED DESCRIPTION OF EMBODIMENTS

It is understood that the subject matter described herein is not limitedto particular embodiments described, as such may, of course, vary. It isalso understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present subject matter is limited onlyby the appended claims. Where a range of values is provided, it isunderstood that each intervening value between the upper and lower limitof that range, and any other stated or intervening value in that statedrange, is encompassed within the disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosed subject matter belongs. Although anymethods and materials similar or equivalent to those described hereincan also be used in the practice or testing of the present disclosedsubject matter, this disclosure may specifically mention certainexemplary methods and materials.

All publications mentioned in this disclosure are, unless otherwisespecified, incorporated by reference herein for all purposes, including,without limitation, to disclose and describe the methods and/ormaterials in connection with which the publications are cited.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present disclosedsubject matter is not entitled to antedate such publication by virtue ofprior invention. Further, the dates of publication provided may bedifferent from the actual publication dates, which may need to beindependently confirmed.

As used herein and in the appended claims, the singular forms “a,” “an”and “the” include plural referents unless the context clearly dictatesotherwise.

Nothing contained in the Abstract or the Summary should be understood aslimiting the scope of the disclosure. The Abstract and the Summary areprovided for bibliographic and convenience purposes and due to theirformats and purposes should not be considered comprehensive.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosed subject matter. Any recited method can be carried out in theorder of events recited, or in any other order that is logicallypossible.

Reference to a singular item includes the possibility that there areplural of the same item present. When two or more items (for example,elements or processes) are referenced by an alternative “or,” thisindicates that either could be present separately or any combination ofthem could be present together, except where the presence of onenecessarily excludes the other or others.

As summarized above and as described in further detail below, inaccordance with the various embodiments of the present invention, thereis provided an apparatus for measuring fluorescent analyte concentrationin the blood and a method for using the apparatus. In some embodiments,the apparatus measures the fluorescent analyte concentrationnoninvasively, e.g., by exciting intact tissue in the patient, e.g., theoral mucosa. In some embodiments, the apparatus measures the fluorescentanalyte concentration by exciting blood samples or other tissue ex vivo.

The apparatus described herein measures the fluorescence of an analyteby excitation of tissue at two alternating wavelengths, or wavelengthranges. The two wavelengths are selected such that the analyte exhibitsa greater difference in fluorescence at the two wavelengths (one ofwhich may be at the excitation peak of the analyte) than that ofbackground fluorophores, and at which blood exhibits substantiallysimilar absorbance at the two wavelengths. The degree of similarity canbe appropriately determined by those of ordinary skill in the art. Tofulfill the requirement of sufficiently similar absorbance of light atthe two excitation wavelengths in tissue, the excitation wavelengths canbe determined experimentally in a way to minimize the effectivepenetration depth differences of light at both wavelengths or bothwavelength ranges. This may be approximated on a representative sample,which does not contain the analyte, by setting the first excitationwavelength at the known wavelength for maximum excitation efficiency ofthe analyte and then scanning the excitation wavelength along the otherside of the blood absorbance peak until the background fluorescencespectrum has the expected intensity and the most similar shape. Anexample of this is provided below.

Although the apparatus is described herein with respect to measurementof eZnPP using the absorbance characteristics of blood (e.g., withhemoglobin as the predominant absorber), it is understood that theprinciples described herein are applicable to measurement of otheranalytes and reference materials having similar fluorescence andabsorbance properties.

eZnPP is an indicator of iron supply to developing red blood cells.During hemoglobin synthesis, if iron deficiency makes iron unavailableto the developing red blood cell to form heme from protoporphyrin IX,then zinc is chelated instead to form eZnPP as one of the firstbiochemical responses to iron depletion.

The measurement of eZnPP by noninvasive tissue excitation requires aquantitative method to distinguish the fluorescence of eZnPP from thatof other fluorophores in tissue, i.e., from tissue autofluorescence.Since eZnPP is found inside erythrocytes only, it has been observed thatthe “dilution” of the blood by tissue that shows autofluorescence doesnot in first order destroy the quantitative nature of the derivedsignal. That is, measurement of eZnPP is insensitive to theconcentration of blood within the tissue, i.e., to the value of theblood volume fraction of the tissue, over a certain range. This range ofinsensitivity to the blood volume fraction can be modified by changingthe probe head configuration, i.e., by changing the spatial separationbetween excitation and detection fiber(s).

In an exemplary embodiment, the fluorometer noninvasively measures eZnPPfluorescence in red blood cells by examination of the microcirculationof the intact oral mucosa at the two alternating excitation wavelengths.The fluorometer can also be used to noninvasively examine other tissue,such as other mucosal surfaces, as well as the skin if permitted by theamount of skin pigmentation. The fluorometer illuminates the mucosa andtransmits the induced fluorescence to a photodetector. Diode lasers maybe used as the excitation light sources. The fluorometer can also beused to examine tissue samples or blood samples ex vivo.

An exemplary embodiment of the fluorometer 100 is shown schematically inFIG. 1. Fluorometer 100 includes an excitation light source 102 forilluminating the tissue T of the patient, and a light detector 104, suchas a spectrophotofluorometer, for analyzing the fluorescence. In someembodiments, two or more detectors are employed, in which part of thesedetectors receive light through optical filters transmitting light inthe wavelength range of the emission wavelength range of the analyte andthe other part of these detectors receive light through optical filterstransmitting light in the wavelength range outside the emission maximumof the analyte. The fluorometer 100 measures the concentration of eZnPPfound in erythrocytes E in the blood vessel V of the patient. Aprocessor 106 determines the concentration of eZnPP based on thefluorescence detected by the detector 104.

The provision of light to the tissue T and transmission of fluorescenceto the light detector 104 is performed by an optical probe head 108 inan exemplary embodiment. The excitation light source 102 providesradiation for tissue excitation at two wavelengths. In an exemplaryembodiment, alternating wavelengths are provided by first and secondlight sources 110 and 112, such as lasers or LEDs which operate at twowavelengths, e.g., 407 nm and 425 nm. A beam combiner 114 provides thelight to the tissue T as a single source in an alternating fashion. Thefrequency of the alternation is chosen quickly enough to show intensityvariations during the measurement, e.g., due to movement of the patient,in both emission spectra in such a way that the variations are reducedor canceled in the difference of the spectra. It has been observed thatthere are high intensity variations if the patient moves duringmeasurement. However, if the speed of the alternation is sufficientlyrapid, these variations are discernible in both emission spectra, and bysubtracting them, the variations are canceled out. It has also beenobserved that measuring the emission spectra in parallel (e.g., with aCCD detector such that all wavelengths are measured simultaneously)avoids the result in which intensity variations due to movement becomewavelength-dependent intensity variations (“peaks”) in the spectrum. Alens and/or filter 116 can be provided to focus and/or direct the lightto the tissue T. Transmission of light from the probe 108 to the lightdetector 104 is accomplished by one or more optical fibers. A singleoptical fiber is used to illuminate the tissue and also to transport thefluorescence to the detector. A lens 120 and/or filter 118 can beprovided to focus and/or remove noise from the light transmitted to thedetector 104. After accounting for background tissue fluorescence,scatter, path length, geometric and other factors, the processor 106correlates the intensity of the fluorescence to the eZnPP/heme ratio andprovides the result in an output device, such as a display screen 130,speaker 132 or vibrating unit 134. An optional communications component130 is provided in certain embodiments, as will be described in greaterdetail hereinbelow. An optional power supply 122, such as a battery, canbe included in the fluorometer, particularly if it is a portable device.In some embodiments, the fluorometer 100 can be directly connected tothe electrical power supply of the home or institution.

Apparatus 100 is illustrated in FIG. 1A which indicates the opticalfiber 111 associated with the light source 102, also referred to hereinas the excitation fiber, and the optical fiber 109 associated with thelight detector, such as spectrometer 104, also referred to herein as thedetection fiber. FIG. 1B illustrates a first spacing between excitationfiber 111 and a detection fiber 109, i.e., “interfiber spacing.” In FIG.1B, the interfiber spacing d=0. FIG. 1C illustrates a second interfiberspacing, i.e., a spacing d of about 1200 μm. The interfiber spacing d inprobe 108 is selected, e.g., experimentally, to obtain minimaldependence on the blood volume fraction over a physiological relevantrange. A photograph of an exemplary embodiment of a portion of thefluorometer is represented in FIG. 2, adapted for measurement of patientblood samples, although such apparatus may be used for tissuemeasurements as well.

In measurements of a blood sample on a glass slide, eZnPP is one of themajor fluorophores with a dominant, characteristic excitation peak atabout 425 nm (FIG. 3) and an emission peak at about 590 nm (FIG. 4).Within the erythrocyte, eZnPP is bound to hemoglobin. Hemoglobin doesnot fluoresce but strongly absorbs light at 400 to 430 nm.

This absorption by hemoglobin diminishes the eZnPP fluorescence. With afront-face hematofluorometer, the hemoglobin and eZnPP in a blood sampleon a glass slide absorb almost all the excitation light within a thinsurface layer that allows the emitted light to be collected with equalefficiency. The intensity of the emission at 590 nm is proportional tothe eZnPP/heme molar ratio.

FIG. 13 compares measurements of patient blood samples (diluted to 4%)taken by the fluorometer 100 (excitation at 425 nm; emission at 590 nm;measurements in a.u., arbitrary units) in the free-beam instrumentalconfiguration shown in FIG. 2 on the vertical axis, with those by anAviv hematofluorometer, on the horizontal axis. Overall, themeasurements are closely correlated; the remaining scatter may beexplained by a greater specificity for eZnPP with the fluorometer 100compared to the Aviv hematofluorometer.

In contrast to measurement of blood samples on a glass slide, innoninvasive measurement of erythrocytes within an examined tissue, e.g.,in the microcirculation of the oral mucosa, eZnPP is a minorfluorophore. Instead, connective tissue (collagen and elastin) is theprincipal source of autofluorescence from the stromal layers containingthe microcirculation. In the thin overlying epithelium ofnon-keratinized oral mucosa (mucous membranes of the lip, buccal andsublingual mucosa), the dominant fluorophores are mitochondrial reducednicotinamide adenine dinucleotide (NADH) and mitochondrial flavinadenine dinucleotide (FAD). The absorbance spectrum of NADH does notextend up to 425 nm and, accordingly, will not contribute tofluorescence at the 590 nm emission peak of erythrocyte eZnPP. As willbe discussed below, the contribution of epithelial FAD to fluorescenceat 590 nm can likely be minimized or eliminated by optimizing theconfiguration of the excitation and detection fibers in the probe head108.

In FIG. 5, an exemplary fiber-optic probe head 108 is illustrated. Acentral detection fiber 109 is surrounded by one or more (e.g., six)excitation fibers 111. FIG. 6 is a schematic diagram of non-keratinizedoral mucosa. The excitation light (indicated by arrow L) must passthrough a thin overlying epithelial layer EL with light-scatteringelements to reach erythrocytes E in the microcirculation in the stromallayer S, also with light-scattering elements.

Using the fiber-optic probe head design disclosed in FIG. 5, the tissueautofluorescence spectrum from the mucous membrane of the lower lip of ahuman patient is shown in FIG. 7. Because the magnitude of the tissueautofluorescence is considerably greater than the fluorescence oferythrocyte eZnPP, some investigators have concluded that measurement inthe oral mucosa is not feasible. See, e.g., Chen X. “Feasibility testfor non-invasive detection of zinc protoporphyrin in oral mucosa andretina.” Biomedical Engineering 2007; M.S.: 1-71.

The fluorometer overcomes limitations in the prior art by providing analternating two-wavelength fluorescence excitation method that is usedto distinguish eZnPP fluorescence from tissue autofluorescence. As shownin FIG. 8, the hemoglobin absorbance is the same at the excitation peakfor erythrocyte eZnPP (425 nm) and at 407 nm. By alternating excitationat two wavelengths, i.e., about 407 and about 425 nm, the fluorescenceemission spectrum excited at 407 nm can be subtracted from that excitedat 425 nm to obtain a difference measurement that is proportional to theeZnPP/heme molar ratio. In some embodiments that excitation occurs attwo wavelength ranges, i.e., about 405 to about 415 nm and about 420 nmto about 430 nm. In a tissue measurement, the intensities of theexciting light sources would be adjusted to give the same fluorescenceemission intensities for autofluorescence. Such adjustment would dependon the light source being used by the apparatus, e.g., whether a laseror some other light source is being used. Also, the emission intensitiescan be normalized (e.g., scaled to the 407 nm emission spectrum). Theresulting difference spectrum would be zero at the point ofnormalization. In the presence of eZnPP, the emission intensity onexcitation at 425 nm would be greater than that at 407 nm. Thedifference of the two emission intensities would be virtually specificfor eZnPP and would depend linearly on the concentration in the targetvolume.

The second excitation wavelength (around 407 nm) excites protoporphyrinIX (“ePP”) fluorescence more efficiently than at 425 nm. Accordingly,information about zinc protoporphyrin (eZnPP and ePP) fluorescence canbe gathered simultaneously from the difference spectrum.

EXAMPLE

A simple tissue phantom for oral mucosa (illustrated in FIG. 9) consistsof an overlying, diffusely scattering film to model the epithelial layerand—to model the stromal layer—a solution containing dissolved elastin,lipofundin as a light-scattering agent, and also a whole blood sample ata 1% dilution (eZnPP 60 μmol/mol heme, the upper limit of normal). FIG.10 shows the fluorescent properties of the tissue phantom, displayingthe excitation spectra for collagen with and without blood. The verticallines indicate excitation wavelengths of 407 and 425 nm.

The results are summarized in FIGS. 11 and 12. Each of the figuresillustrates F_(m) (emitted fluorescence) at the indicated excitationwavelength. Simple measurement of the emission spectra at 590 nm, inaccordance with conventional techniques, would be unable to detect anemission peak for eZnPP (FIG. 11). By contrast, FIG. 12 illustrates theuse of alternating two-wavelength fluorescence excitation. Accordingly,the difference spectrum F_(m)(425 nm)−F_(m)(407 nm) clearly shows thecharacteristic emission peak at 590 nm for erythrocyte eZnPP in thistissue phantom whose concentration is at the upper limit of the normalrange. In whole blood, the output is automatically quantitative for theratio C(eZnPP)/C(Hemoglobin). Without being bound to a particulartheory, it is understood that as long as the optical scattering insidethe tissue doesn't vary significantly, the output will be quantitativeas well. However, additional considerations include intra-/inter-patientvariations and/or probe head geometry.

Fluorometer 200 is illustrated in FIG. 14 and is generally identical tofluorometer 100 discussed above, with the substantial differences notedherein. In an exemplary embodiment, the fluorometer 200 is a portableunit that includes a power supply (not shown), such as a watch-typebattery or a rechargeable battery. The fluorometer 200 may include ahousing 226 in which the detector, the processor and the power supplyare housed. In order to provide portability, the housing 226 may have anoverall length of about 2 inches to 6 inches. Fluorometer 200 includes aprobe 208 used to illuminate the tissue being examined, e.g., the mucosaor a blood sample, and transmit the fluorescence to a light detector bydepressing an activation switch 228.

The fluorometer 200 also includes one or more output components. In someembodiments, the output component is disposed in or on the housing.Exemplary output components include a display screen 230, a speaker 232,and/or a vibrating component (not shown). The display screen 230 may bean LCD display, an AMOLED display or the like. The output of theparticular output components may be used to signal to the user that theanalyte reading was successfully completed. For example, the displayscreen 230 may display an icon that is illuminated when successfulanalyte readings are obtained. The speaker 232 can provide an audiblesignal that the analyte reading was obtained successfully. The vibratingcomponent similarly can provide a vibration signal to indicatesuccessful analyte readings. Such tactile or audible outputs areparticularly useful if the analyte reading is self-administered, or ifthe testing is performed in settings where bright sunlight or otherconditions make it difficult to view the display screen.

The output component further provides an indication of the concentrationof analyte in the tissue being examined. In some embodiments, thedisplay provides a numerical indication of the analyte concentration240. The speaker 232 may alternatively, or in addition, provide thenumerical analyte concentration audibly.

For certain users of the fluorometer 200, the raw analyte informationmay not be meaningful. Accordingly, the fluorometer 200 may allow theuser or a health care provider to enter threshold concentrations for ahealth range of analyte concentration. In some embodiments, thethreshold concentrations may be entered at the time of manufacturing,e.g., programmed in software or hard-coded. In use, the fluorometer 200would determine whether the detected concentration of iron is below apreselected concentration of iron. Such concentration of iron may beselected based on the circumstances, e.g., to determine whether anindividual is iron-deficient or iron-replete. The display or otheroutput device would provide an indication 242 to the user that the ironconcentration was below this threshold. For example, the display wouldprovide an indication “LOW” iron concentration or “IRON REPLETE” etc.The speaker audibly provides the same phrase. A vibrating component canbe programmed to vibrate in a certain manner to indicate that athreshold has been exceeded, e.g., two consecutive vibrations for lowiron concentrations.

The fluorometer 200 may also be programmed to track trends in theanalyte concentration over time. In some embodiments, the fluorometer200 includes a memory that stores multiple analyte readings, which canbe tagged with a patient identification and a time stamp. Whensuccessive analyte readings are obtained for a particular patient, thefluorometer 200 can determine whether the analyte concentrations areincreasing or decreasing as well as the rate of such increase ordecrease. Trend indications 244 are provided by the display, e.g.,upward or downward trend arrows or alphanumeric indications such as“IRON CONCENTRATION INCREASING” or “IRON CONCENTRATION DECREASING.” Thespeaker 230 and vibrating component can likewise provide suchinformation to the user in a similar manner as described above for theanalyte concentration.

Fluorometer 300 is generally identical to fluorometer 100 discussedabove, with the substantial differences noted herein. In someembodiments, it is useful to provide a separate monitor unit 360 thatallows the user to obtain information remotely from the patient. Thefluorometer 300 includes a communications component for communicatingwith the monitor unit 360. The fluorometer 300 can include a wiredconnection to the monitor, e.g., by use of a USB connection. Asillustrated in FIG. 15, the communications component may be wirelesslyconnected to the monitor and may include an RF transmitter, IRtransmitter, Bluetooth transmitter or a WiFi transmitter for providingthe detected eZnPP concentrations or other information about the patientor the fluorometer to a receiver on the monitor unit 360. Communicationsbetween the fluorometer and monitor may be achieved by providing acellular transmitter (GSM, CDMA, etc.) or satellite transmitter on thefluorometer.

The communications component can provide signals to the monitorunit—signals that relate to the analyte concentration. Suchcommunications may occur immediately or at a predetermined time aftertaking the analyte reading of the patient. In some embodiments, thefluorometer 300 may transmit the analyte reading when the analyteconcentration is determined to exceed a threshold.

The monitor unit 360 includes a receiver that receives the signal fromthe fluorometer 300. In the case of a wireless transmission, the monitorcan include an RF, IR, Bluetooth or WiFi receiver. For a wiredconnection, the receiver component 360 may include the electricalcontacts for the wired connection. The monitor unit 360 also includes aprocessor, memory component, power supply and user interface. Thedisplay screen may be omitted from the fluorometer 300 in someembodiments.

A user interface is provided on the monitor unit 360, which can includea display unit 330, speaker 332, vibrating component, and input controls336, e.g., switches, buttons, soft keys, keyboard, touch screeninterface and the like. The user interface can provide an indication ofthe concentration of analyte in the tissue 340. The user interfaceprovides an indication that the concentration of analyte exceeds apredetermined threshold 342, e.g., by indicating that the ironconcentrations are “LOW.”

The memory provided on the monitor unit 360 stores successive analyteconcentrations and can provide an indication 344 that the concentrationof analyte is increasing or decreasing from the previous concentrationof analyte, e.g., with trend arrows.

The monitor unit 360 can be programmed, e.g., through the user interfaceor by factory settings, to store health goals for the patient. Suchhealth goals include overall fitness concentrations and may include atarget analyte concentration, e.g., achieving a recommended ironconcentration within a desired time frame. The monitor unit 360 canevaluate whether the patient is reaching the health goal. For example,the monitor may determine that the patient's iron concentrations areincreasing. The user interface may then provide an indication to thepatient that the trend of iron concentrations is towards the health goaland that the user has attained 50% of the patient's health goal with abar graph-type display 346. In some embodiments, the display screen 330may provide an icon that changes color (e.g., from red to green) orincreases in size as an indication that iron concentrations areimproving. The speaker 332 can audibly provide the same information.

The user interface can provide a treatment suggestion to the patientafter determining the analyte concentration and/or comparing the analyteconcentration to the patient's health goals. For example, the userinterface may provide a suggestion for consuming a nutritionalsupplement to address the analyte concentration, e.g., consumption ofiron-rich foods or supplementation, and the quantity of suchsupplementation 348. The user interface may suggest the patient take apharmaceutical compound to address the particular analyte concentration.

The monitor unit 360 can be a fixed component in a clinical setting. Insuch case, the monitor unit can be a desktop or laptop personal computerand receive power by an AC household current. In some embodiments, themonitor unit 360 can be a portable unit. For example, the monitor can bea laptop computer, a cellular telephone, a tablet computer or the like.The monitor can be a portable dedicated handheld unit.

Fluorometer 400 is illustrated in FIGS. 16 and 17, and is identical tofluorometer 100 discussed above, with the differences noted herein. Inan exemplary embodiment, the fluorometer 400 includes a “free beam”configuration (e.g., a fluorometer without a fiber-optic probe). It isunderstood that fluorometer 400 can alternatively include a fiber-basedconfiguration. In an exemplary embodiment, fluorometer 400 distinguishesiron deficient blood samples from iron replete blood samples. Whentesting is performed in vitro, the sample is tested with or without thepresence of an agent to mimic light scattering in tissue. When testingis performed in vivo, the light source is applied to the intact patienttissue, e.g., the oral mucosa, as discussed hereinabove. An embodimentof the instrumentation is shown schematically in FIGS. 17 and 18.

Fluorometer 400 includes an excitation light source 402 for illuminatingthe tissue T of the patient and a light detector 404 for analyzing thefluorescence. In some embodiments, the light source 402 is a 500 Wshort-arc Xe-lamp (T-light. Karl Storz, Tuttlingen, Germany) white lightsource, and the light detector 404 is a cooled CCD spectrometer. Thefluorometer 400 measures the concentration of eZnPP found inerythrocytes in vivo in the blood vessel of a patient, or in vitro in ablood sample T maintained in a cuvette 405. The in vitro measurementscan be performed on diluted blood samples, with a concentration of 2%whole blood in phosphate buffered saline. The sample volume can be about3000 μl, including 60 μl whole EDTA blood in a cuvette. As discussedabove regarding fluorometer 100, a processor (not shown) determines theconcentration of eZnPP based on the fluorescence detected by thedetector 404.

The provision of light to the tissue T and transmission of fluorescenceto the light detector 404 is performed by excitation light source 402,which provides radiation for tissue excitation. In an exemplaryembodiment, alternating wavelengths are provided by a tunable opticalfilter 440. In some embodiments, the optical filter unit consists of thefilter 440, as described in greater detail herein, with a tunablewavelength and a tunable bandwidth, a detection unit that can detectemission spectra from 520 to 1000 nm, and incorporates a free beamformat that can be readily converted to a fiber-based configuration. Inthe testing configuration, light was optically filtered such that thetransmitted light's central wavelength was tunable in the bluewavelength range, 395 nm to 431 nm, while preserving a spectralbandwidth h (e.g., 5 nm full width/half maximum, “FWHM”). Light in thewavelength range 500 nm-750 nm was suppressed with OD>10.

The fluorometer 400 implements the techniques described herein formeasurement of a fluorescent analyte using alternating wavelengths (407and 425 nm) for tissue excitation. Procedures were established forprotoporphyrin measurements in whole blood samples using the referenceHPLC method (Immundiagnostik AG) and the conventional front-facehematofluorometer 452 (Aviv; shown in FIG. 17), and theeZnPP-fluorometer 400.

With continued reference to FIG. 16, a collimating lens 415 and/orclean-up filter 417 can be provided to focus and/or direct the light tothe sample T via a dichroic beam splitter 419 and lens 421. The bluelight beam was focused onto the sample T, with a focus diameter of 2 mm.On the sample T, the total excitation light power was 6 mW (centralwavelength 425 nm, wavelength-dependent). The fluorescence light emittedfrom the sample T was transmitted backwards through the beam splitter419 and filtered by a long-pass filter 418 (e.g., OG515, Schott AG,Mainz, Germany) and lens 420, limiting the usable detection range to 520nm-750 nm. Finally, the fluorescence light was coupled into across-section converting fiber 409, consisting of seven 200 μm-diameteroptical fibers, arranged in a circle. These fibers, linearly arranged atthe other end of the fiber bundle, were coupled into a temperatureregulated CCD spectrometer 404 (e.g., detection range: 340 nm-1022 nm,S2000-TR, Ocean Optics, Inc., Dunedin, Fla., USA), yielding an effectivespectral resolution of 5 nm.

To optionally allow correction for wavelength- and time-dependentintensity variations, fluorescence standard measurements were performed.For example, the emission intensity of the short-arc lamp 402 iswavelength dependent, and the filtered light's intensity is alsowavelength dependent. Also, the total power of the lamp 402 may changeduring usage. The fluorescence standard includes a 1 mm thick piece ofcommercially available solid polymethyl methacrylate containing RhodaminB (1BF/RB, Starna GmbH, Pfungstadt, Germany), fixed at the wall of thecuvette 405.

After accounting for background tissue fluorescence, scatter, pathlength, geometric and other factors, the processor correlates theintensity of the fluorescence to the eZnPP/heme ratio and provides theresult in an output device, such as a display screen, speaker orvibrating unit. An optional communications component is provided incertain embodiments, as will be described in greater detail hereinbelow.An optional power supply, such as a battery, can be included in thefluorometer, particularly if it is a portable device. In someembodiments, the fluorometer 400 can be directly connected to theelectrical power supply of the home or institution.

Tunable Filter

The instrumentation of tunable optical filter 440 is illustrated inFIGS. 16 and 18. The filter unit 440 allows the simultaneous selectionof both the central filtered wavelength and the spectral bandwidth, foruse in applications requiring detection of light or illumination bylight at a small spectral bandwidth. The tunable optical filter unit 440provides improved light transmission efficiency and makes possiblespectral filtering of images and fiber bundles without a scanningdevice. Although the filter unit 440 is described herein in connectionwith fluorescence spectroscopy, the filter unit 440 can find applicationin fluorescence microscopy, fluorescent imaging, and in advancedmicroscopy applications such as fluorescence-lifetime imagingmicroscopy. For illumination, the filter 440 can be used with filteredincoherent light sources to achieve strong illumination intensities atsmall spectral bandwidths, such as illumination for fluorescencemicroscopy, fluorescence spectroscopy, and more generally, inapplications in which a tunable laser is not practical, e.g., due tocost.

As illustrated in FIG. 18, filter unit 440 includes two tunable bandpassoptical filters 444 and 446, e.g., Semrock Versachrome filters capableof independent rotation for selecting an angle of incidence with a beamof light passing through the filter unit 440. After acquiring thefluorescence emission spectra for the excitation wavelengths, shutter442 was closed to prevent further illumination of the sample T. A darkspectrum can be recorded with the same settings. Step motor controller448 is provided to allow independent rotation of the two step motorscarrying the optical filters at a high rotation speed with sub-degreeprecision.

The components, design and function of the filter unit 440 are shownschematically in FIGS. 19-24. In FIG. 19, a beam of light passingthrough filter unit 440 is designated as beam portions 480, 482, and484. Beam portion 480, a collimated beam of unfiltered light typicallyhaving a large spectral width, is transmitted through optical filter444, e.g., a Versachrome filter capable of rotation as indicated in theangular direction of arrow R1 (as well as in the opposite angulardirection indicated by arrow R1). The filtered light beam 482, filteredby filter 444, has a fixed spectral bandwidth, and the centralwavelength can be selected by the angle of filter 444 with respect tothe incidence of beam 480 on filter 444. The light beam 482 istransmitted through filter 446, e.g., a Versachrome filter capable ofrotation as indicated in the angular direction of arrow R2 (as well asin the opposite angular direction indicated by arrow R2), with theresulting light beam 484. The results are illustrated in FIG. 20,showing the proportion of effective transmission through the two filters444 and 446. The solid lines represent transmission through the firstfilter 444 for two different angles defined with respect to thedirection of the light beam. Typically, angle 2 is greater than angle 1.The dotted lines represent the transmission through the second filter446 for two different angles. The total transmission characteristics ofthe filter unit 440 are the product of both transmission curves (filter444 and filter 446), solid and dotted, shown as a shaded area under theoverlap of the curves in FIG. 20. In the exemplary embodiment, filter444 and filter 446 provide about 60% transmission of light. If twosimilar filters are used for filters 444 and 446, the angles of the twofilters have to be chosen independently because the angular dependencyof the filter transmission is non-linear. Example: Angle 1: a(Filter444)=20°, a(Filter 446)=0° resulting in a 5 nm spectral bandwidth; Angle2: a(Filter 444)=40°, a(Filter 446)=35° also resulting in a 5 nmspectral bandwidth at a lower central wavelength.

As shown in FIG. 21, after transmission of the light through filters 444and 446, a beam splitter 419 can be added to the filter unit 440 formonitoring a part of the filtered light, e.g., light beam 586. This partof the light can be detected, e.g., by a spectrometer 404 or power meterto monitor the spectral bandwidth, the power or both.

As shown in FIGS. 22-25, during non-perpendicular transmission of alight beam through a filter, a parallel offset of the light beam occurs.If the filters are arranged as shown in FIG. 22, the offset provided byfilters 444 and 446 accumulates to a large net offset. As illustrated inFIG. 22, the offset O1 of light beam 682 with respect to light beam 680,and the offset O2 of light beam 684 with respect to light bean 682results in a total offset 03 with respect to light beam 680. (A dashedline is used to represent the hypothetical trajectory of light beam 680in the absence of filters 444 and 446.) If the filters are rotatedcounter-wise, i.e., in opposite directions, as shown in FIG. 23, theoffset O6 of light beam 684′ with respect to light beam 680′ (thecumulative offset of offset O4 and offset O5) is significantly reducedfrom offset O3 but not eliminated because the angles of both filters 444and 446 are different. In some embodiments, as illustrated in FIG. 24, athird optical element 449, e.g., a planar piece of glass that provides100% transmission and refractive index n>1, can be inserted, in whichcase the offset O10 between light beam 680″ and light beam 686″ iseliminated (i.e., O10=0).

During in vitro testing, blood samples and tissue phantoms may contain alight-scattering agent to mimic the optical properties of tissue such asthe oral mucosa. Latex microspheres having a diameter of about 0.5 μmwere found to have little autofluorescence and remained suspended duringthe time required for testing, and thus are a suitable, exemplarylight-scattering agent.

FIGS. 25-30 illustrate the excitation and emission spectra of tissue.The z-axis represents arbitrary units (a.u.). A single excitationcentral wavelength, with a bandwidth of 5 nm full width at half maximum(FWHM), was used to obtain the associated emission spectra. To determinethat 407 and 425 nm are the optimal alternating wavelengths formeasurements of blood samples, excitation-emission matrices wereobtained in blood samples (2% in saline) with eZnPP concentrations inthe reference (“normal”) range of 30 to 80 μmol/mol heme and irondeficient range (>80 μmol/mol heme), as determined by conventionalfront-face Aviv hematofluorometer. (In the Figures, “ZnPP” refers to theerythrocyte zinc protoporphyrin concentration. In FIG. 25, for example,“ZnPP=198” means erythrocyte zinc protoporphyrin concentration of 198μmol ZnPP/mol heme.) Typical matrices are shown in FIGS. 25-30. TheeZnPP peak 804 is illustrated in FIGS. 25-30 at excitation of 425 nm.For the determination of eZnPP levels in blood, 407 nm and 425 nmprovide the optimal performance as the alternating wavelength pair.

As shown in FIGS. 31-36, the alternating (407 nm-425 nm) wavelengthmethod significantly reduces or eliminates the background whole bloodautofluorescence that produces an elevated baseline with conventionalsingle wavelength (425 nm) studies. The y-axis represents arbitraryunits (a.u.). The conventional single wavelength measurement (425 nm) isindicated by line 802 (upper line) and its associated eZnPP peak 804.(FIG. 31 corresponds to the data illustrated in FIG. 25 for anexcitation wavelength of 425 nm and eZnPP=75.) The alternating (407nm-425 nm) wavelength method in accordance with the embodimentsdescribed herein is indicated by line 806 with an associated eZnPP peak808.

EXAMPLE

A study was performed using 35 anonymous patient whole blood samplesfrom the Institut für Laboratoriumsmedizin, Klinikum der UniversitatMunchen, which were analyzed prospectively for erythrocyte zincprotoporphyrin (“eZnPP”) concentration by the reference HPLC method(Immundiagnostik AG), by the Aviv hematofluorometer, and by theZnPP-fluorometer 400 in a free beam configuration as described herein.

The reference (“normal”) range of eZnPP concentrations was 30 to 80μmol/mol heme. The iron deficient range of eZnPP concentrations was >80μmol/mol heme. The study included eZnPP-fluorometer measurements of theblood samples (i) without a light scattering agent, with a blood volumefraction of 0.02, (ii) with the smaller (0.5 μm) latex microspheres as ascattering agent to provide scattering coefficients over a physiologicrange (reduced scattering coefficient about μ_(s)′=1 to 4 mm⁻¹), incombination with (iii) a physiologic range of blood volume fractions(about 0.02 to 0.08). In aggregate, results were obtained for a seriesof studies of the 35 blood samples under 11 different combinations oflight scattering and whole blood concentrations over the physiologicrange for each of the 35 blood samples. In these samples, the prevalenceof iron deficiency, as determined by the HPLC reference method, was 69%.

For all measurements, the light source was tuned to 425 nm and 407 nm(central wavelengths) with a spectral bandwidth of 5 nm FWHM. Afteracquiring the fluorescence emission spectra for the excitationwavelengths, a shutter was closed to prevent further illumination of thesample, and a dark spectrum was recorded with the same settings.

For the measurement of the Rhodamin B fluorescence standard, the CCDspectrometer's integration time was set to 40 ms, averaging internallyover 16 spectra. Including the time required for the wavelength-tuningof the filter unit and the shutter, the measurement time was 4 s. Forthe measurements of blood samples, which showed much dimmerfluorescence, the integration time was set to 400 ms, averaginginternally over 4 spectra, resulting in a total measurement time of 10s. It was verified that during measurements the signal remained stable.

An exemplary spectral calibration and normalization process is describedherein. From all raw, uncorrected spectra F_(uncorrected)(λ) thecorresponding dark spectrum D(λ) was subtracted. The resulting spectrumwas multiplied by the factor C_(excitation) which depends on theexcitation wavelength and is used to compensate for wavelength- andtime-dependent excitation light intensity variations, as well as foroptical adjustment variations. In addition, the resulting spectra weredivided by the wavelength-dependent transmission of the detection filterT_(filter)(λ) and multiplied by a wavelength-dependent factor includingoptical fiber transmission and spectrometer sensitivityC_(spectrometer)(λ). These additional calibration factors allowcomparing the corrected spectra F_(corrected)(λ) to spectra that weremeasured using other devices, because influences of the spectralsensitivity of the detection optics and the spectrometer arecompensated. The complete calibration procedure is shown in Equation(1):

$\begin{matrix}{{F_{corrected}(\lambda)} = {\left\lbrack {{F_{uncorrected}(\lambda)} - {D(\lambda)}} \right\rbrack \frac{C_{spectrometer}(\lambda)}{T_{filter}(\lambda)}C_{excitation}}} & (1)\end{matrix}$

To obtain C_(excitation), at first the calibration procedures describedabove (dark subtraction, filter transmission, detection sensitivitycalibration, except for the factor C_(excitation)) were applied also tothe fluorescence standard measurement F_(RhodaminB)(λ). Then,C_(excitation) was calculated as described in Equation (2): The “real”value of the Rhodamin B fluorescence maximum from a referencemeasurement, maxR_(RhodaminB)(λ) was divided by the maximum of theRhodamin B fluorescence measured by the prototype measurement set-upmaxF_(RhomodaminB)(λ). The reference measurement was recorded by afluorescence spectrometer (Fluoromax-2, Jobin Yvon GmbH, Unterhaching,Germany), with excitation and detection monochromator adjusted to matchexcitation and detection bandwidth (5 nm FWHM) of the measurementset-up.

$\begin{matrix}{C_{excitation} = \frac{\max \mspace{14mu} {R_{RhodaminB}(\lambda)}}{\max \mspace{14mu} {F_{RhodaminB}(\lambda)}}} & (2)\end{matrix}$

For purposes of comparison with the novel two wavelength excitationmethod described herein, a method is described herein which requires oneexcitation wavelength band (e.g., centered at 425 nm) and two emissionwavelength bands (centered at 573 nm and 593 nm) (also referred to as“two wavelength emission method.”). For in vitro testing, a samplemeasurement of patient blood (HPLC determined eZnPP/heme ratio=333μmol(ZPP)/mol(heme) and PP/heme ratio=605 μmol(PP)/mol(heme)) is shownin FIG. 37. Using the reference HPLC method, the eZnPP and PPconcentrations C_(ZPP) and C_(PP) were determined as absoluteconcentrations in units (nmol/l). Separately, the hemoglobinconcentration C_(Heme) was determined by standard laboratory tests inunits (g/dl). From the HPLC determined eZnPP and hemoglobinmeasurements, C_(ZPP) and C_(Heme), the eZnPP/heme ratio (and in thesame way, from C_(PP) and C_(Heme) the PP/heme ratio) is calculated byEquation (3), using the hemoglobin subunit's molecular weight 64,458g/mol.

$\begin{matrix}{{\frac{C_{ZPP}}{C_{Heme}}\left\lbrack \frac{\lambda \; {mol}}{mol} \right\rbrack} = {{\frac{C_{ZPP}\left\lbrack {{nmol}\text{/}l} \right\rbrack}{C_{Heme}\left\lbrack {g\text{/}{dl}} \right\rbrack}\frac{64\text{,}458}{10^{4}}} = \frac{C_{ZPP}\left\lbrack {{nmol}\text{/}l} \right\rbrack}{0.1551 \cdot {C_{Heme}\left\lbrack {g\text{/}{dl}} \right\rbrack}}}} & (3)\end{matrix}$

In FIG. 37, a calibrated fluorescence emission spectrum F₄₂₅ in thewavelength range 520 nm-750 nm is shown (solid line in FIG. 37). Theemission maximum of eZnPP is at 593 nm; the background fluorescence fromblood plasma F_(Background) was fitted as an exponential decay curve tothe data in the spectral range (dashed line in FIG. 37), withoutcontribution from porphyrin emission.

With continued reference to FIG. 37, the eZnPP fluorescence intensityI_(ZPP) was calculated according to Equation (4) below. The measuredfluorescence intensities were taken from the calibrated emissionspectrum by averaging over the wavelength range 590 nm-596 nm (I₅₉₃) andover 570 nm-576 nm (I₅₇₃). The background fluorescence intensity at 593nm I_(bkg,593) (double arrows, dashed line) cannot be measured directly,but can be calculated from the fluorescence intensity at 573 nm (I₅₇₃),e.g., 0.8 times the fluorescence intensity at 573 nm. The difference ofI₅₉₃ and I_(bkg,593) was used to quantify the eZnPP/heme ratio.

I _(ZPP) =I ₅₉₃ −I _(bkg,593) =I ₅₉₃−0.8·I ₅₇₃   (4)

According to one aspect of the disclosed subject matter, a novelevaluation method is described herein for reducing the influence ofbackground fluorescence on the detected intensity at 593 nm (alsoreferred to as “two wavelength excitation method.”) The method uses twoexcitation wavelength bands, e.g., 407 and 425 nm. For quantification ofthe eZnPP/heme ratio, one emission wavelength band, centered at 593 nm,is used. In addition, the ePP/heme ratio is quantified by a secondemission wavelength band centered at 627 nm.

The method is illustrated in FIG. 38, in which two corrected emissionspectra (F₄₂₅; solid line in FIG. 38 and F₄₀₇; dot-dashed line in FIG.38; left axis description) of the patient blood sample described aboveare shown; the central excitation wavelengths were 425 nm and 407 nm,respectively. For optimized overlap in the 520 nm-570 nm region, F₄₀₇was scaled by a factor 1.15. Additionally, the difference between thesespectra is shown, which is referred to as “difference spectrum.”

In FIG. 38, the differences of the two spectra at 593 nm and at 627 nmare illustrated by dashed line (right axis description) and highlightedby arrows: As the excitation wavelength 407 nm approaches the PPexcitation maximum at 397 nm and is far off the eZnPP excitation maximumat 424 nm, the emission spectrum F₄₀₇ shows a pronounced PP fluorescenceemission peak, which is found at 627 nm, compared to the lower eZnPPfluorescence peak at 593 nm. The difference in the range 520 nm-570 nmbecomes nearly zero, which shows that the background fluorescence iseliminated by calculating the difference spectrum.

The difference spectrum is then used to evaluate both eZnPP and PPfluorescence. The eZnPP/heme ratio can be directly quantified byevaluating the fluorescence intensity at 593 nm (averaging over 590nm-596 nm), as illustrated in FIG. 39. Also, a eZnPP emission spectrumis drafted. At 627 nm, a linear combination of eZnPP and PP fluorescenceintensities yields the signal at 627 nm: the eZnPP fluorescence(positive value in the difference spectrum) and the PP fluorescence(negative value in the difference spectrum). Accordingly, a measure forthe PP/heme ratio hp can be calculated according to Equation (5): The PPfluorescence is the negative of the detected fluorescence at 627 nm /₆₂₇plus the eZnPP fluorescence intensity at 627 nm, which equals ⅓ of theeZnPP fluorescence intensity at 593 nm.

I _(PP) =−I ₆₂₇+(⅓)I ₅₉₃   (5)

The eZnPP/PP ratio was calculated by dividing the eZnPP/heme and thePP/heme ratios, I_(ZPP)/I_(PP). As both ratios are given in arbitraryunits, this calculated eZnpp/PP ratio is also given in arbitrary units.

MATLAB (R2010a, MathWorks®, Natick, Mass., USA) was used for statisticaldata evaluation. For the statistical evaluation of the correlation oftwo methods, a linear regression was calculated using a least square fit(function polyfit) and the Pearson product-moment correlationcoefficient (PCC) R-value was calculated (function corrcoef) as well asthe p-value (e.g., test against the hypothesis of no correlation, tstatistics, correlation if p<0.05).

Results of the testing described herein are illustrated in FIGS. 40-47.The eZnPP/heme ratio's correlation of the Aviv hematofluorometer and thereference standard HPLC is shown in FIGS. 40-41. The error bars indicatethe precision of each method, being 9% (HPLC) and 15%(hematofluorometer). The linear regression is shown in FIG. 40 (solidblack line). The number of samples n=35, the correlation coefficientR=0.967, with a p-value p<0.0001. The relative residuals of themeasurement and the linear regression are shown in FIG. 41, being in therange −0.39 . . . +0.46.

The precision of the eZnPP peak intensity evaluation of the fluorescencespectroscopic measurements was 10%, determined by repeated measurementsof the same sample. The correlation of the eZnPP fluorescence intensityat 593 nm evaluated by the two wavelengths emission method (y-axis) andthe eZnPP/heme ratio (μmol eZnPP/mol heme) measured by HPLC measurements(x-axis) is shown in FIGS. 42-43. The error bars indicate the precisionof each method for three exemplary measurements. The linear regressionis shown in FIG. 42 (solid black line). The number of samples n=35, thecorrelation coefficient R=0.978, with a p-value p<0.0001. The relativeresiduals of the measurement and the linear regression are shown in FIG.43, being in the range −0.48 . . . +0.53.

The precision of the evaluated eZnPP peak intensity of the differencespectrum was 10%, determined by repeated measurements of the samesample. The correlation of the eZnPP fluorescence intensity at 593 nmevaluated by the two wavelengths excitation method (y-axis) and theeZnPP/heme ratio (μmol eZnPP/mol heme) measured by HPLC measurements(x-axis) is shown in FIGS. 44-45. The error bars indicate the precisionof each method for three exemplary measurements. The linear regressionis shown in FIG. 44 (solid black line). The number of samples n=35, thecorrelation coefficient R=0.976, with a p-value p<0.0001. The relativeresiduals of the measurement and the linear regression are shown in FIG.45, being in the range −0.50 . . . +0.57.

The precision of the PP peak intensity, calculated from the intensity ofthe difference spectrum at 627 nm and 593 nm using Equation (5), was15%, determined by repeated measurements of the same sample. Thecorrelation between the measured PP fluorescence intensity of thedifference spectrum at 627 nm evaluated by the two wavelengthsexcitation method (y-axis) and the PP/heme ratio (μmol PP/mol heme)measured by HPLC measurements (x-axis) is shown in FIGS. 46-47. Theerror bars indicate the precision of each method for three exemplarymeasurements. The linear regression is shown in FIG. 46 (solid blackline). The number of samples n=35, the correlation coefficient R=0.996,with a p-value p<0.0001. The relative residuals of the measurement andthe linear regression are shown in FIG. 47, being in the range −0.37 . .. +0.50.

The eZnPP/PP ratio, calculated from the eZnPP and PP fluorescenceintensities for each patient blood sample (n=35), is shown in FIG. 48.The average eZnPP/PP ratio was 2.74 (arbitrary units), with a range 0.64. . . 7.91 (arbitrary units).

It is also understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting, since the scope of the present subject matter is limitedonly by the appended claims.

1-109. (canceled)
 110. An apparatus for noninvasive simultaneousmeasurement of a concentration of erythrocyte zinc protoporphyrin(eZnPP) and erythrocyte protoporphyrin IX (ePP) as the eZnPP/heme ratioand the ePP/heme ratio in the blood of a patient comprising: a lightsource operatively associated with a tunable optical filter, such thatthe light source is adapted to excite the tissue of the patient atalternating first wavelength and second wavelength ranges, wherein thefirst wavelength range is selected to encompass an eZnPP excitation peakand the second excitation wavelength range is selected so that theabsorbance of blood is similar to that of the first excitationwavelength range; one or more spectrometers for detecting a portion ofthe emission spectrum of the eZnPP and the ePP excited at the firstexcitation wavelength and the second excitation wavelength; and aprocessor adapted to determine a derived signal representative of theconcentration of the eZnPP and the ePP based on the difference betweenportions of the emission spectra excited at the first excitationwavelength range and the second excitation wavelength range anddetermine whether the detected concentration of the eZnPP or the ePP isbelow a preselected concentration; and provide an indication that theeZnPP or the ePP concentration is below the preselected concentration.111. The apparatus of claim 110 wherein the light source is adapted toprovide the first wavelength range centered at about 425 nm and thesecond wavelength range centered at about 407 nm.
 112. The apparatus ofclaim 110 wherein the processor quantifies the eZnPP/heme ratio based onthe emission wavelength band centered at about 593 nm.
 113. Theapparatus of claim 110 wherein the processor quantifies the ePP/hemeratio based on the emission wavelength band centered at about 627 nm.114. The apparatus of claim 110 wherein the processor quantifies theeZnPP/heme ratio based on the difference of intensity between theemission spectrum excited at about 425 nm and the background emissionspectrum excited at about 407 nm, wherein the difference is quantifiedat a wavelength band centered at about 593 nm.
 115. The apparatus ofclaim 110 wherein the processor quantifies the ePP/heme ratio based on alinear combination of eZnPP and PP fluorescence centered at about 627 nmas the difference between the emission spectrum intensity of the eZnPPand the detected fluorescence, compared to the background emissionspectrum.
 116. The apparatus of claim 110, wherein the tunable filterunit comprises a first optical filter and a second optical filter, thefirst and second optical filters capable of independent variation of theangle of incidence of light provided by the light source.
 117. Theapparatus of claim 110, wherein the emission spectra of the eZnPP andePP define a wavelength range, and wherein the detector includes one ormore light sensitive elements receiving light through the one or moreoptical filters transmitting light in the wavelength range of theemission spectra of the eZnPP and ePP.
 118. The apparatus of claim 110,further comprising a probe comprising an optical fiber associated withthe light source and an optical fiber associated with the one or morespectrometers.
 119. The apparatus of claim 118, wherein interfiberspacing of the optical fiber associated with the light source and theoptical fiber associated with the one or more spectrometers is selectedsuch that the derived signal is insensitive to the blood volumefraction.
 120. The apparatus of claim 118, wherein interfiber spacing ofthe optical fiber associated with the light source and the optical fiberassociated with the one or more spectrometers is selected to achieve amaximum detection sensitivity at a selected depth of the tissue. 121.The apparatus of claim 120, wherein the selected depth of the tissue isselected as the depth having the highest expected concentration ofeZnPP.
 122. The apparatus of claim 110, further comprising at least oneadditional unit selected from a memory unit, an output component, acommunications component, and a monitor unit.
 123. A method fornoninvasive simultaneous measurement of a concentration of erythrocytezinc protoporphyrin (eZnPP) and erythrocyte protoporphyrin IX (ePP) asthe eZnPP/heme ratio and the ePP/heme ratio in the blood of a patientcomprising: exciting the tissue at alternating first wavelength andsecond wavelength ranges, the first and second excitation wavelengthranges selected such that the first wavelength range is selected toencompass an eZnPP excitation peak and the second excitation wavelengthrange is selected so that the absorbance of blood is similar to that ofthe first excitation wavelength range and the emission spectra excitedat the first and second excitation wavelength ranges exhibit adifference in emission intensities is greater than that of backgroundfluorophores and light absorbance by blood is similar; detecting aportion of the emission spectra excited at the first excitationwavelength range and the second excitation wavelength range; and using aprocessor, determining the concentration of the eZnPP and the ePP basedon the difference between the emission spectra excited at the firstexcitation wavelength range and the second excitation wavelength range,determining whether the detected concentration of the eZnPP or the ePPis below a preselected concentration, and providing an indication thatthe eZnPP or the ePP concentration is below the preselectedconcentration.
 124. The method of claim 123 wherein first wavelengthrange is centered at about 425 nm and the second wavelength range iscentered at about 407 nm and wherein the processor quantifies theeZnPP/heme ratio based on the emission wavelength band centered at about593 nm the processor quantifies the ePP/heme ratio based on the emissionwavelength band centered at about 627 nm.
 125. The method of claim 123wherein the processor further determines whether the eZnPP or the ePPconcentrations are increasing or decreasing when successive analytereadings are obtained for a particular patient and provides anindication that the eZnPP or the ePP concentration is increasing ordecreasing.
 126. A system for noninvasive simultaneous measurement of aconcentration of erythrocyte zinc protoporphyrin (eZnPP) and erythrocyteprotoporphyrin IX (ePP) as the eZnPP/heme ratio and the ePP/heme ratioin the blood of a patient comprising: the apparatus of claim 1; and atleast one additional component selected from a memory unit, an outputcomponent, a communications component, and a monitor unit.
 127. Thesystem of claim 126, wherein the memory unit stores multiple eZnPP orePP readings, wherein the apparatus is configured to determine whetherthe eZnPP or the ePP concentrations are increasing or decreasing whensuccessive readings are obtained for a particular patient and provide anindication that the eZnPP or the ePP concentration is increasing ordecreasing.
 128. The system of claim 126, wherein the memory unit storeshealth goals for a patient wherein the apparatus is configured tocompare the detected eZnPP or ePP concentration to a targetconcentration and provide an indication whether the target concentrationis reached.
 129. The system of claim 126 wherein the apparatus isconfigured to provide a treatment suggestion to a patient afterdetermining the concentration of eZnPP or ePP.